It is known that in a development or differentiation process, a sugar-chain structure reflects a cell state, and thus, is changed. Therefore, a considerable number of differentiation markers or cancer markers, which are now widely used, recognize a sugar chain. For example, a stage specific embryonic antigen 1 (SSEA1) that is a differentiation marker for a developmental state is an antibody to a sugar-chain structure, which is called Lewis X: Galβ1, 4GlcNAc (Fucα1,3)-, and the epitope of CA19-9 that is used as a colon cancer marker for a medical clinical service is a sugar-chain antigen, which is called sialyl Lewis A: SAα2, 3Galβ1, 3GlcNAc (Fucα1,4)-. The sugar chain of the cell surface as described above sensitively reflects a type of cell and a differentiation stage, and thus, it is easy to be a very available candidate for a biomarker.
As a method for detecting a disease-specific sugar-chain change, lectins, which are a sugar-chain binding protein derived from a plant or fungus, have been used along with an anti-sugar-chain antibody for a long time. When a tissue slice is stained with lectins, it is possible to separately stain the cells having different properties or the cells having different differentiation states, but since the sugar-chain recognizing specificity of the lectin is not clear, it is difficult to specify what kind of the sugar-chain structure is being modified. It is known that wisteria floribunda lectin (Wisteria floribunda agglutinin, WFA) that is one of plant lectins belongs to Leguminosae lectins, and recognizes a sugar chain including N-acetylgalactosamine (GalNAc) residue. However, the detailed specificity thereof is not clear. Nevertheless, the unique sugar-chain recognition specificity of WFA is used as a marker in various biological fields. For example, in the field of neuroscience, it is known that WFA is a classical marker for staining perineuronal network (PNN) (Non Patent Literature 1), and WFA also stains a normal foveolar epithelial cells of normal gastric mucosa (Non Patent Literature 2). It is also used in the identification method for identifying a prostate cancer and prostatic hypertrophy (Patent Literature 1). In addition, recently, the effectiveness of WFA as a biomarker that is used for diagnosis is highlighted, and thus, it is reported that WFA-positive MUC1 is a bile marker for diagnosing intrahepatic cholangiocarcinoma (Patent Literature 2 and Non Patent Literature 3).
The isolations of the lectin from wisteria floribunda seeds are reported by a plurality of groups in the 1970s (Table 1).
TABLE 112345autherToyoshimaToyoshimaKurosawaCheungKaladasS.S.T.G.P. M.year1971 1975 1976 1979 1979 namemitogenhemag-agglu-hemag-mitogenicglutinintininglutininlectinm.w.3234322832KDa(mono)m.w.67136 685766KDa116 (oligo)235 oligomerdimertetramerdimerdimerdimertetrameroctamerRef. (4) (5) (6) (7) (8)
Toyoshima, and others report the isolations and biochemical analysis of two kinds of glycoprotein lectins having different molecular weights (WFM and WFH). Wisteria floribunda Mitogen (WFM) that forms the dimer of 67 KDa, in which the molecular weight of monomer is about 32 KDa, has hemagglutinating activity and phytogen activity (Non Patent Literature 4), but Wisteria floribunda hemagglutinin (WFH) of 136 KDa that is the tetramer of 35 KDa monomers does not have mitogen activity, but has strong hemagglutination activity and leukoagglutination activity as compared with WFM (Non Patent Literature 5). Meanwhile, the lectin purified by Kurokawa is a 68 KDa glycoprotein formed by the S—S bond of two 32 KDa subunits, and has hemagglutination activity inhibited by GalNAc (Non Patent Literature 6). For these two groups, each of the lectins is purified by a conventional biochemical isolation method of a protein, but for the group of Poretz and others, the homodimer lectin (Non Patent Literature 7) formed by the S—S bond between 28 KDa monomers having hemagglutination activity and the lectin having mitogen activity (66 KDa dimer formed by 32 KDa monomers) are isolated by the affinity to polyleucyl hog gastric mucin (Non Patent Literature 8). These lectins derived from wisteria floribunda seeds that are reported until now have similar property, such as, GalNAc recognition, but have subtle distinctions for amino acid compositions, sugar compositions, molecular weights, and the like. Therefore, it is difficult to determine whether or not the molecules are the same.
The WFA has grown in biological importance, but the sugar-chain recognition of WFA is not yet clear, and also, it is unclear whether the sugar-chain structures recognized by the WFAs in the neuron and stomach are the same or not. In addition, for the production of WFA, the WFAs that are sold by Vector Laboratory Company or EY Laboratory Company as a reagent are purified from natural wisteria floribunda seeds. Therefore, in order for the stable supply or in order to manage the uniformity among purification lots, it is required to shift the production thereof to the recombinant lectin production by a genetic engineering technique.